pharmaceutics
Article123 I-BMIPP, a Radiopharmaceutical for Myocardial Fatty Acid

Metabolism Scintigraphy, Could Be Utilized in Bacterial
Infection Imaging
Yuka Muranaka 1 , Asuka Mizutani 2 , Masato Kobayashi 2 , Koya Nakamoto 1 , Miki Matsue 3 , Fumika Takagi 4 ,Kenichi Okazaki 4 , Kodai Nishi 5 , Kana Yamazaki 6 , Ryuichi Nishii 6 , Naoto Shikano 7 ,Shigefumi Okamoto 2,8 , Hideki Maki 4 and Keiichi Kawai 1,9, *
1

2

3

4

5

6

Citation: Muranaka, Y.; Mizutani, A.;

7

Kobayashi, M.; Nakamoto, K.;
Matsue, M.; Takagi, F.; Okazaki, K.;

8

Nishi, K.; Yamazaki, K.; Nishii, R.;
et al. 123 I-BMIPP, a9Radiopharmaceutical for Myocardial
Fatty Acid Metabolism Scintigraphy,
Could Be Utilized in Bacterial
Infection Imaging. Pharmaceutics
2022, 14, 1008. https://doi.org/
10.3390/pharmaceutics14051008
Academic Editor: Leonard I. Wiebe
Received: 18 March 2022
Accepted: 3 May 2022
Published: 7 May 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.

*

Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, 5-11-80 Kodatsuno,
Kanazawa 920-0942, Japan; yukarisa93@stu.kanazawa-u.ac.jp (Y.M.); kou.nakamoto@sage.ocn.ne.jp (K.N.)
Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University,
5-11-80 Kodatsuno, Kanazawa 920-0942, Japan; mizutani.a@staff.kanazawa-u.ac.jp (A.M.);
kobayasi@mhs.mp.kanazawa-u.ac.jp (M.K.); sokamoto@mhs.mp.kanazawa-u.ac.jp (S.O.)
Ishikawa Prefectural Institute of Public Health and Environmental Science, 1-11, Taiyogaoka,
Kanazawa 920-1154, Japan; mmiikkii_0804@pref.ishikawa.lg.jp
Laboratory for Drug Discovery & Disease Research, Shionogi & Co., Ltd., 3-1-1, Futaba-cho,
Toyonaka 561-0825, Japan; fumika.takagi@shionogi.co.jp (F.T.); kenichi.okazaki@shionogi.co.jp (K.O.);
hideki.maki@shionogi.co.jp (H.M.)
Department of Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto,Nagasaki 852-8523, Japan; koudai@nagasaki-u.ac.jp
Department of Molecular Imaging and Theranostics, Institute for Quantum Medical Science,
Quantum Life and Medical Science Directorate, National Institutes for Quantum Science and Technology,
4-9-1 Anagawa, Inage, Chiba 263-8555, Japan; yamazaki.kana@qst.go.jp (K.Y.); nishii.ryuichi@qst.go.jp (R.N.)
Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami,
Inashiki 300-0394, Japan; sikano@ipu.ac.jp
Advanced Health Care Science Research Unit, Innovative Integrated Bio-Research Core Institute for Frontier
Science Initiative, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan
Biomedical Imaging Research Center, University of Fukui, 23-3 Matsuoka-shimoaizuki, Eiheiji-cho,
Yoshida-gun, Fukui 910-1193, Japan
Correspondence: kei@mhs.mp.kanazawa-u.ac.jp; Tel.: +81-76-265-2527; Fax: +81-76-234-4366

Abstract: In this study, we evaluated the use of 15-(4-123 I-iodophenyl)-3(R,S)-methylpentadecanoic
acid (123 I-BMIPP) to visualize fatty acid metabolism in bacteria for bacterial infection imaging. We
found that 123 I-BMIPP, which is used for fatty acid metabolism scintigraphy in Japan, accumulated
markedly in Escherichia coli EC-14 similar to 18 F-FDG, which has previously been studied for bacterial
imaging. To elucidate the underlying mechanism, we evaluated changes in 123 I-BMIPP accumulation
under low-temperature conditions and in the presence of a CD36 inhibitor. The uptake of 123 I-BMIPP
by EC-14 was mediated via the CD36-like fatty-acid-transporting membrane protein and accumulated
by fatty acid metabolism. In model mice infected with EC-14, the biological distribution and wholebody imaging were assessed using 123 I-BMIPP and 18 F-FDG. The 123 I-BMIPP biodistribution study
showed that, 8 h after infection, the ratio of 123 I-BMIPP accumulated in infected muscle to that incontrol muscle was 1.31 at 60 min after 123 I-BMIPP injection. In whole-body imaging 1.5 h after 123 IBMIPP administration and 9.5 h after infection, infected muscle exhibited a 1.33-times higher contrast
than non-infected muscle. Thus, 123 I-BMIPP shows potential for visualizing fatty acid metabolism ofbacteria for imaging bacterial infections.
Keywords: 123 I-BMIPP; SPECT; nuclear medicine imaging; bacterial infection; bacterial imaging

This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Bacteria exhibiting antimicrobial resistance (AMR), such as fluoroquinolone-resistantEscherichia coli and penicillin-resistant Streptococcus pneumoniae, have emerged due to thePharmaceutics 2022, 14, 1008. https://doi.org/10.3390/pharmaceutics14051008

https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2022, 14, 1008

2 of 10

inappropriate use of antimicrobial agents [1–3]. It is predicted that, as the number of bacteria
resistant to existing antimicrobial agents increases, conventional treatment methods will
no longer be applicable, and treatment will thus become more difficult. The World HealthOrganization has advocated the development of international AMR countermeasure actionplans detailing the investigation and monitoring of AMR trends, the appropriate useof antimicrobial agents, and research and development strategies for new antimicrobial
agents [4,5].
With the increasing awareness of the seriousness of infectious diseases involving AMR,
molecular imaging techniques have attracted attention for bacterial infection imaging. Nuclear medicine imaging is particularly useful as a new non-invasive diagnostic method thatcan be used to identify the site of an infection and follow up on the effects of treatment [6,7].
Various radiopharmaceuticals have been investigated for bacterial infection imaging. The
development and application of radiopharmaceuticals, such as 99m Tc-based derivatives,
99m Tc-labeled antibiotics, 11 C-labeled D-amino acids, and 2-deoxy-2-[18 F]fluoro- D -glucose(18 F-FDG), which target components of the bacterial cell wall, are still in progress [6,8].
These compounds include not only carbon sources (D-glucose), nitrogen sources (aminoacids), and minerals but also fatty acids, which are essential for bacterial growth [9,10].
However, to date, no radiopharmaceutical for bacterial infection imaging that targets fatty
acid metabolism has been developed. We examined the use of 15-(4-123 I-iodophenyl)3(R,S)-methylpentadecanoic acid (123 I-BMIPP), a radiopharmaceutical commonly used insingle-photon emission computed tomography (SPECT) myocardial fatty acid metabolism
scintigraphy in Japan, for bacterial imaging in comparison with 18 F-FDG, which is alreadywidely used in imaging.
2. Materials and Methods
123 I-BMIPP was purchased from Nihon Medi-Physics Co., Ltd. (Tokyo, Japan). 18 F-

FDG was synthesized at the PET facility of Kanazawa University.
2.1. Bacterial Strain and Culture Conditions
Escherichia coli EC-14 was used as the clinical isolate strain. EC-14 was obtained from
Shionogi & Co., Ltd. (Osaka, Japan). For pre-cultivation, stock EC-14 in 50% glycerol
was mixed with THY medium consisting of Todd-Hewitt Broth (Becton, Dickinson andCo., Franklin Lakes, NJ, USA) and 0.2% yeast extract (Becton, Dickinson and Co.) at aratio of 1:100. EC-14 was incubated at 37 ◦ C for 12–14 h with shaking. Subsequently,
EC-14 (1.2 × 108 colony-forming units (CFU)/100 µL) was seeded in amino-acid-freeDulbecco’s Modified Eagle’s Medium (DMEM; Fujifilm Wako Pure Chemical Corporation,
Osaka, Japan; 048-33575) and incubated at 37 ◦ C with shaking. Bacterial protein wasmeasured using a Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA,
USA; 23227).
2.2. Accumulation of Radiopharmaceuticals in E. coli EC-14
EC-14 (1.2 × 108 CFU/100 µL) was seeded in 5 mL of amino-acid-free DMEM andincubated for 1, 3 and 6 h. After incubation, 37 kBq/10 µL of 123 I-BMIPP and 18 F-FDG
was added and incubated for 5 min at 37 ◦ C with shaking. For in vitro experiments using
18 F-FDG, the concentration of glucose in the medium was adjusted to 0.1 mg/mL, sinceamong the three glucose concentrations examined (4.5, 1.0 and 0.1 mg/mL), the highest
accumulation of 18 F-FDG was observed at a concentration of 0.1 mg/mL.
EC-14 was collected by centrifugation at 7000× g for 10 min at 4 ◦ C and then washedthree times with 5 mL of phosphate-buffered saline (PBS; Medical & Biological Laboratories
Co., Ltd., Aichi, Japan). Samples were suspended in 1 mL of 0.1 M NaOH and measured
using a gamma counter (AccuFLEX ARC-γ7010, Aloka Medical, Tokyo, Japan).

Pharmaceutics 2022, 14, 1008

3 of 102.3. Accumulation of 123 I-BMIPP in E. coli EC-14 under Low-Temperature Conditions
EC-14 (1.2 × 108 CFU/100 µL) was seeded in 5 mL of amino-acid-free DMEM andincubated under ice-cold conditions at 4 ◦ C for 3 h. After incubation, 37 kBq/10 µL of
123 I-BMIPP was added to the solution, and they were incubated for 5 min at 4 ◦ C withshaking. The radioactivity accumulated in the EC-14 was then measured as described in
the Section 2.2. Accumulation of Radiopharmaceuticals in E. coli EC-14.
2.4. Accumulation of 123 I-BMIPP in E. coli EC-14 in the Presence of a CD36 Inhibitor
EC-14 was cultured under the same conditions as described in Section 2.2. Accumulation of Radiopharmaceuticals in E. coli EC-14. After incubation at 37 ◦ C, 37 kBq/50 µL of
123 I-BMIPP and 50 µL of 1.0 mM sulfosuccinimidyl oleate (SSO, Cayman Chemical, Ann
Arbor, MI, USA), an inhibitor of CD36, were mixed and added to the cell culture. CD36
binds long-chain fatty acids and promotes their transport into cells. The radioactivity
accumulated in EC-14 was measured using the same method as described in the Section 2.2.
2.5. Mouse Model of E. coli EC-14 Infection
All experiments were conducted in accordance with the ethical standards of ouruniversity (Animal Care Committee of Kanazawa University, AP-183983) and with international standards for animal welfare and institutional guidelines. EC-14 was cultured in
Luria–Bertani broth (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) for 12 h andthen seeded into new broth and incubated at 37 ◦ C for 12–14 h with shaking. Subsequently,
EC-14 was collected by centrifugation at 8000× g for 5 min at 4 ◦ C and suspended for
inoculation into mice. Male Jcl:ICR mice (n = 4) (4 weeks old; CLEA Japan, Tokyo, Japan)
were purchased 7 days prior to the experiments and subjected to immunosuppression
treatment with 150 mg/kg and 100 mg/kg of endoxan (Shionogi) at 4 days and 1 day prior
to infection, respectively.
EC-14 (approximately 5 × 106 CFU/100 µL) was injected into the muscle of the hindleg of mice under anesthesia. Mice were anesthetized using a mixture of butorphanol
tartrate, midazolam, and medetomidine hydrochloride (Fujifilm Wako Pure Chemical
Corp., Osaka, Japan). Mice were euthanized at 2, 6, 8 and 24 h after infection. Subsequently,
the infected muscle was collected and homogenized in PBS. The number of CFU in thehomogenized samples was determined by dilution in PBS and plating on agar medium.
2.6. Biological Distribution of 123 I-BMIPP and 18 F-FDG in E. coli EC-14 Infection Model Mice
EC-14 (approximately 5 × 106 CFU/100 µL) was injected into the muscle of the hindleg of mice (n = 4) as described in the Section 2.5. At 2 and 8 h after infection, mice were
injected intravenously with 370 kBq/110 µL of 123 I-BMIPP and 10–17 MBq/90 µL of 18 FFDG. The mice injected with 123 I-BMIPP and 18 F-FDG were different individuals. Before
administration, the mice were fasted for 4 h. Mice were euthanized at 15 and 60 min
after administration, and blood, heart, lung, liver, kidney, infected perineal muscle, andcontralateral non-infected perineal muscle (control muscle) tissues were collected. The
collected organs were dissolved by adding 1.0 mL of solubilizer, Solvable (PerkinElmer,
Waltham, MA, USA), and crushed with a disposable homogenizer, BioMasher® (Nippi,
Tokyo, Japan, 49118-71). These samples and initial 123 I-BMIPP- and 18 F-FDG-associatedradioactivity were measured using a gamma counter (AccuFLEX ARC-8001; Hitachi Aloka
Medical, Tokyo, Japan). In heart, lung and kidney, the radioactivity and weight of wholeorgans were measured. In liver, a part of the liver was cut out, and the radioactivity andweight were measured. Weight of whole liver was also measured, and the radioactivity ofwhole liver was calculated using radioactivity of a part of the liver. Data are reported aspercent injected dose per gram of tissue (%ID/g).

2.7. Planar Imaging with 123I-BMIPP in E. coli EC-14 Infection Model Mice
Pharmaceutics 2022, 14, 1008

At 8 h post-infection, 123I-BMIPP (18.5 MBq/300 µL/mouse) was injected into the tail4 of 10vein of EC-14 infection model mice under lead shielding from head to bladder (n = 3).
Imaging conditions were set as follows: matrix, 512 × 512 pixels; magnification, 100%. The
mice were anesthetized,
and a gamma camera (MiniCam, Inter Medical, Lübbecke, Ger2.7. Planar Imaging with 123 I-BMIPP in E. coli EC-14 Infection Model Micemany) was used to image 123
the mice.
At 8 h post-infection,
I-BMIPP (18.5 MBq/300 µL/mouse) was injected into the
Planar image was acquired for 5 min (1 frame) at 1.5 h after 123I-BMIPP administration
tail vein of EC-14 infection model mice under lead shielding from head to bladder (n = 3).
and9.5 hconditions
after EC-14
infection.
Imagesmatrix,
were analyzed
data analysis
Imaging
were
set as follows:
512 × 512 using
pixels;AMIDE
magnification,
100%. software(ver.
1.0.4).
The mice were anesthetized, and a gamma camera (MiniCam, Inter Medical, Lübbecke,
Germany) was used to image the mice.

2.8. Statistical
Analysis
Planar image
was acquired for 5 min (1 frame) at 1.5 h after 123 I-BMIPP administration
and Data
9.5 h after
EC-14 infection.
Imagesandwerestandard
analyzed deviation
using AMIDE
analysisusing
softwareare presented
as means
anddataanalyzed
the F-test(ver. 1.0.4).

and Student’s t-test. All analyses were conducted using GraphPad Prism 8 statistical software(GraphPad
Software, Inc., La Jolla, CA, USA). A p value of less than 0.05 was con2.8. Statistical
Analysissideredindicativeof a asstatisticallydifference.
Data
are presented
means and significant
standard deviation
and analyzed using the F-test andStudent’s t-test. All analyses were conducted using GraphPad Prism 8 statistical software3.(GraphPad
Results Software, Inc., La Jolla, CA, USA). A p value of less than 0.05 was considered
indicativeof a statisticallysignificant difference.
3.1.
Accumulationof Radiopharmaceuticals
in E. coli EC-14

Table 1 shows the accumulation of 123I-BMIPP and 18F-FDG in EC-14 5 min after each
3. Results
123I-BMIPP accumulated in EC-14 at 6.65%ID/ng protein at 1 h of incubation,
addition.
3.1. Accumulationof Radiopharmaceuticals in E. coli EC-14
3.52%ID/ng
protein3 h and 2.68%
ID/ng
proteinatF-FDG
6 h. 18in
F-FDG
in EC-14
Table 1 showstheataccumulationof 123
I-BMIPPand 18EC-14accumulated
5 min after each
123 I-BMIPP
ataddition.
6.32%ID/ng
proteinat 1 h of incubation,
15.9%ID/ngprotein
proteinand 11.8%ID/ng
accumulated
in EC-14 at 6.65%ID/ng
at at
1 h3ofhincubation,
123I-BMIPP
3.52%ID/ng
proteinat 3 h and
2.68%
ID/ng
at 6 h. 18of
F-FDG
accumulated
in EC-14proteinat 6 h.
At a culturetimeof 1h,
the protein
accumulation
was higher
than that18
proteinitatwas
1 h lowerof incubation,
15.9%ID/ng
h and11.8%ID/ng of 123Iofat 6.32%ID/ng
F-FDG, whereas
at culture
times of 3protein
and 6ath.3The
accumulation
123
protein in
at EC-14
6 h. At5amin
culturetimeof 1h, the
was
than
BMIPP
after
injection
andaccumulation
the growth ofcurveI-BMIPP
of EC-14
arehigher
shown
in Figure
18
that
of
F-FDG,
whereas
it
was
lower
at
culture
times
of
3
and
6
h.
The
accumulation
123
1. 123I-BMIPP exhibited marked accumulation in E. coli EC-14 during the early growthof
I-BMIPP in EC-14 5 min after injection and the growth curve of EC-14 are shownphase.
123

in Figure 1.
I-BMIPP exhibited marked accumulation in E. coli EC-14 during the early
growth phase.
Table 1. Accumulation of radiopharmaceuticals in E. coli EC-14.
Table 1. Accumulation of radiopharmaceuticals in E. coli EC-14.

Culture Time(h)Culture Time (h)
1
3 1
6 3
6

Accumulation (%ID/ng Protein)
18F-FDG
Accumulation (%ID/ng Protein)
123I-BMIPP
18 F-FDGConcentration)(0.1 mg/mL Glucose
123 I-BMIPP(0.1 mg/mL Glucose
Concentration)
6.65 ± 0.49
6.32 ± 2.31
6.65 ± 0.49
6.32 ±
3.52 ± 0.20
15.9
± 2.31
1.37
3.52 ± 0.20
15.9 ± 1.37
2.68 ± 0.23
11.8 ± 0.49
2.68 ± 0.23

%ID/ngprotein indicates percent injected dose per ng of protein.
%ID/ng protein indicates percent injected dose per ng of protein.

11.8 ± 0.49

Figure 1. Accumulation of 123 I-BMIPP in E. coli EC-14 5 min after addition. EC-14 was incubated in

Figure 1. Accumulation of 123I-BMIPP in E. coli EC-14123
5 min after addition. EC-14 was incubated inamino-acid-free DMEM for 1, 3 and 6 h. Accumulation of I-BMIPP
is greatest 1 h after incubation,amino-acid-free DMEM for 1, 3 and 6 h. Accumulation of 123I-BMIPP is greatest 1 h after incubation,
during the early logarithmic growth phase.
during the early logarithmic growth phase.

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3.2. Accumulation of 123I-BMIPP in E. coli EC-14 under Low-Temperature Conditions

The accumulation of 123I-BMIPP in E. coli EC-14 under low-temperature conditions issummarized in Figure
2. The accumulation rate was 0.28-fold lower than that of the con123I-BMIPP in E. coli EC-14 under Low-Temperature Conditions
3.2. Accumulation
Accumulation ofof 123
3.2.
I-BMIPP in E. coli EC-14 under Low-Temperature
trol incubated at 37 °C.
123I-BMIPP in E. coli EC-14 under low-temperature conditions is
The accumulation
accumulationof
of123
The
low-temperature conditions issummarizedinFigure2.
Theaccumulationrate
ratewaswas
0.28-fold
lower
than
that
of theconsummarized in Figure 2. The accumulation
0.28-fold
lower
than
that
of thecontrol◦trol incubated
37 °C.
incubated
at 37atC.

Figure 2. Accumulation of 123I-BMIPP in E. coli EC-14 under low-temperature conditions. EC-14 was
123I-BMIPP in E. coli EC-14 under low-temperature conditions.
123I-BMIPP
Figure 2.
2. Accumulation
Accumulationof 123
EC-14
was
Figureof
I-BMIPP E.
coli EC-14
low-temperature
EC-14
incubated
at 4 °C in amino-acid-freeDMEM
for 3 h. Afterincubation,conditions.
waswasadded and
123I-BMIPP was added andincubated at
atinamino-acid-freeamino-acid-free
DMEMforfor3rate
3h.h.After
After
incubation,
incubated
Cmin
in
DMEM
incubation,
I-BMIPP wasand lower
incubated
for445◦°Cat
4 °C. The accumulationof EC-14
at low 123
temperature
is addedsignificantly
incubated
fortheminat 44 ◦°C.
accumulation
rate ofof EC-14
at low
is significantly
significantly lower
lower
incubatedfor
55 minat
C.
The
accumulation rate
EC-14 at
low temperaturetemperature is
thanthat ofcontrolatThe
37 °C.
than thatthat ofof thethe control
controlat
at37
37◦°C.
thanC.

123I-BMIPP in E. coli EC-14 in the Presence of a CD36 Inhibitor
3.3. Accumulation of123
123

3.3. Accumulation
Accumulation ofof I-BMIPP
3.3.
I-BMIPP in
in E.
E. colicoli EC-14
EC-14 inin thethe Presence
Presence ofof aa CD36
CD36 Inhibitor
Inhibitor
123I-BMIPP in E. coli EC-14 incubated in the
Figure3summarizes
theaccumulation
of
123
123
Figure3summarizes
theaccumulation
of
I-BMIPP
in
E.
coli
EC-14
incubated inin thetheFigure 3 summarizes the accumulation of I-BMIPP in123E. coli EC-14 incubated
I-BMIPPaccumulated
inatEC-14 atpresence
ofaaCD36
CD36inhibitor.
inhibitor.
Under
these
conditions,123I-BMIPP
123
accumulatedin
EC-14
presence
of
Under
these
conditions,presence of a CD36 inhibitor. Under these conditions, I-BMIPP accumulated in EC-14 at
10.8%ID/ng
proteinat
1 ofhincubation,
2.41%ID/ng
proteinat
3 1.07%ID/ng
h1.07%ID/ng
and 1.07%ID/ng
10.8%ID/ng protein
proteinatat
ofof
incubation,
2.41%ID/ng
proteinat33hhand
andprotein protein
10.8%ID/ng
11 hh
incubation,
2.41%ID/ng
proteinat
proteinat6h.
The
difference
betweenthecontrolandSSO
treatment
at1
hof
incubation
at
6
The
difference
betweenthecontrolandSSO
treatment
at1
hof
incubationwas
notwas not
at 6 h.
difference between the control and SSO treatment at 1 h of incubation was not
123
123
123
significant. At
Atandincubation,
however,theraterate
I-BMIPPaccumulation
wassignificant.
At333andand66 h6h ofhofof
incubation,
however,theofofrateI-BMIPP
of I-BMIPP
accumulation
wassignificant.
incubation,
however,theaccumulation
wassignificantly lower
lower
than
that
of thethethecontrol.
significantly
thatofcontrol.
significantly
lowerthan
than
that
of
control.

Figure 3. Accumulation of 123I-BMIPP in E. coli EC-14 in the presence of sulfosuccinimidyl oleate(SSO), a CD36 inhibitor. EC-14
was incubated at 4 °C in amino-acid-free DMEM for 1, 3 and 6 h.
123123Figure3.3. Accumulation
I-BMIPP
coli
EC-14
in 50
thein
presenceof sulfosuccinimidyl
oleate oleate
123
FigureAccumulation
ofkBq/50
I-BMIPP
inI-BMIPP
E. coliEC-14
thepresencesulfosuccinimidyl
After incubation
at 37 °C,of37
µL in
of E.
and
µL
of 1.0mM
SSOof(CaymanChemical)◦ C in amino-acid-free DMEM for 1, 3 and 6 h.
(SSO),aCD36
inhibitor.
EC-14
was
incubated
at4(SSO),a CD36at 4there
°C in
DMEM
for 1, 3inand 6 h.
were mixed
andinhibitor.
added. AtEC-14
3 h andwas
6 h incubatedof incubation,
is aamino-acid-freestatistically significant
decrease◦ C, 37 kBq/50 µL of 123 I-BMIPP
123I-BMIPP
After
incubation
atat
3737
and and
50 µL50ofµL
1.0of
mM(CaymanChemical)accumulationcomparedwith
Afterincubation°C,
37 kBq/50µLcontrol.
of 123I-BMIPP
1.0SSOmM
SSO (Cayman
Chemical)were mixed
AtAt
3 h3and6 h 6ofhincubation,
therethere
is a statisticallysignificant
decreasedecreaseinweremixedandandadded.
added.
h andof incubation,
is a statisticallysignificant
in
123 I-BMIPP accumulation compared with control.
123
I-BMIPP accumulation compared with control.

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Pharmaceutics 2022, 14, 1008

6 of 103.4. Growth of E. coli EC-14 in Infection Model Mice
3.4. Growth of E. coli EC-14 in Infection Model Mice

Figure 4 shows a growth curve for EC-14 in the leg muscle of model mice. O

Figure 4 shows a growth curve for EC-14 in the leg muscle of model mice. On average,
age, approximately 1.9 × 106 CFU of EC-14 were present in the muscle tissue 2
approximately 1.9 × 106 CFU of EC-14 were present in the muscle tissue 2 h after infection,
10 CFU at 8
infection,
andofthenumberof bacteria
108 atand1.824× h10after
andthe number
bacteria
increased
to 4.9 ×increased
108 and 1.8to× 4.9
1010×CFU
8 and
h after infection,
respectively.
infection,
respectively.

FigureGrowthcurveof E. coli
EC-14
leg muscle
of muscle
infectionof
model
mice. Immunosuppressed
Figure4. 4.
Growthcurveof E.
coli inEC-14
in leginfectionmodel mice. Immunosu
6
6
mice
infected
withwithapproximately
5 × 10 5CFU/100µL of EC-14
in themuscleof thehind of the
µL of
EC-14
in themusclemicewerewere
infected
approximately
× 10 CFU/100
leg.
h afterinfection,
infection, thenumberof EC-14
is approximately
1.9 × 106 1.9
CFU,× increasing
At 2Ath2afterthemeanmeannumberof EC-14
is approximately
106 CFU,toincreasin
10 CFU at 8 and 24 h after infection, respectively.
4.9
108 and
1.8 1.8
× 10×1010CFU
at 8 and 24 h after infection, respectively.
108×and3.5. Biological Distribution of 123 I-BMIPP and 18 F-FDG in EC-14 Infection Model Mice

3.5. Tables
BiologicalDistribution of 123I-BMIPP and 18F-FDG in EC-14 Infection Model Mice
2 and 3 summarize the biological distribution of 123 I-BMIPP and 18 F-FDG,

respectively,model mice.
In comparison
with the accumulationof 18 andF- 18F-F
Tablesin2EC-14
and infection
3 summarize
the biologicaldistributionof 123I-BMIPP
123
FDG
at 2 and 8inh EC-14
after infection,
the accumulationof In I-BMIPP
was higher
the accumulatio
blood,
spectively,
infectionmodel mice.
comparison
withinthelung, liver and kidney.
FDGTableat 24 and8 h after infection, the accumulation of 123I-BMIPP was higher in thsummarizes the accumulation of 123 I-BMIPP and 18 F-FDG in the EC-14-infectedlung, liverand kidney.
muscleand normal,
uninfected muscle (control). The accumulation of both 123 I-BMIPP and
18 F-FDG was higher in the control mice than in the infected mice. 123 I-BMIPP accumulation
tendedto Biologicalbe higher thanthat of 18 F-FDG
at both in
2 and
8 h EC-14
post-infection,
the rateofTable 2.
distributionof 123I-BMIPP
E. coli
infectionand
model
mice.
accumulation in the infected muscle was significantly higher 8 h post-infection. In thisofto123the
I-BMIPP(%ID/g)study, contrast means the ratio Accumulationof the infected area
contralateral
normal area.

Time after Infection (h)28123Time after I-BMIPP Injection
60
15
6
123 15(min) Accumulation of I-BMIPP (%ID/g)
Time after Infection
Blood (h)16.692 ± 0.93 16.10 ± 1.88 813.65 ± 3.57 19.43
Time after 123 I-BMIPP
HeartInjection
29.11 ± 15
4.48 30.3760± 3.90 30.66
1525.86 ± 6.46
60
(min)
Lung
13.02 ± 2.00 11.84 ± 1.02 11.99 ± 1.10 12.01
Blood
16.69 ± 0.93 16.10 ± 1.88 13.65 ± 3.57 19.43 ± 2.91
Liver
3.86± 4.4811.04± 1.92± 6.403.32 12.02
Heart
25.86 ±17.31
6.46 ±29.11
30.37± 3.90 20.84
30.66 ±
Lung
13.02
±
2.00
11.84
±
1.02
11.99
±
1.10
12.01
±
1.65
Kidney14.01 ± 1.30 12.18 ± 1.27 12.00 ± 1.32 11.79

Table 2. Biological distribution of 123 I-BMIPP in E. coli EC-14 infection model mice.

Liver

17.31 ± 3.86

11.04 ± 1.92

20.84 ± 3.32

%ID/g indicatespercent injected 14.01
dose±perof±tissue.
Kidney1.30gram
12.18
1.27 12.00 ± 1.32

12.02 ± 1.91
11.79 ± 1.88

%ID/g indicates percent injected dose per gram of tissue.

Table 3. Biological distribution of 18F-FDG in E. coli EC-14 infection model mice.

Accumulation of 18F-FDG (%ID/g)
Time after Infection (h)218Time after F-FDG Injection
15
60
(min)

8
15

Kidney

5.43 ± 1.95

2.15 ± 0.52

5.77 ± 1.55

2.71

%ID/g indicates percent injected dose per gram of tissue.
Pharmaceutics 2022, 14, 1008

7 of 10

Table 4 summarizes the accumulation of I-BMIPP and F-FDG in the EC
fected muscle and normal, uninfected muscle (control). The accumulation of bo
18 F-FDG in E. coli EC-14 infection model mice.
Table 3. Biologicaldistribution
BMIPP
and 18F-FDG
wasofhigher
in the control mice than in the infected mice. 123I-B
18F-FDG at both 2 and 8 h post-inf
accumulation tended to behigher thanthat ofAccumulationof 18 F-FDG(%ID/g)and therate
ofInfection
accumulationin the infectedmuscle was significantly
higher 8 h p
Time
after
(h)
2
8
fection.
this
study,
contrast means the ratio of the infected area to the contralater
18 F-FDG
TimeInafter
Injection
15
60
15
60
(min)
mal area.
123

18

Blood
2.83 ± 0.90
0.69 ± 0.09
0.93 ± 0.11
0.18 ± 0.03
123
18
Heart
13.95
±
5.47
15.05
±
3.35
30.02
±
7.33
28.57
± 5.98mice.
Table 4. Accumulation of I-BMIPP and F-FDG in E. coli EC-14 infection model
Lung
2.91 ± 0.76
2.96 ± 1.04
3.44 ± 0.39
4.04 ± 0.88
Liver2.95 ± 1.07
1.10
± 0.262.81 ± 0.49
2.10 ± 0.65
123
18F-FDG
I-BMIPP
After In5.43 ± 1.95
2.15 ± 0.52
5.77 ± 1.55
2.71 ± 0.67
After Infec- Kidney

jectionAccumulation%ID/g indicates percent
injected dose per gram of tissue.

Accumulation
Con
(min)
(%ID/g)(%ID/g)Table 4. Accumulation of 123 I-BMIPP and 18 F-FDG in E. coli EC-14 infection model mice.
Infected
6.09 ± 3.72
1.69 ± 0.50
15
1.15 18
1
123 I-BMIPP
F-FDG
Control
5.30 ± 0.72
1.61 ± 0.53
2 (min)After Injection
Accumulation 2.47 ± 0.69
Accumulation 6.13 ± 1.14
InfectedContrast
Contrast
60
1.00
0(%ID/g)(%ID/g)Control
6.10 ± 1.03
2.54 ± 0.81
Infected
6.09 ± 3.72
1.69 ± 0.50
1.15
15
Infected
4.251.05± 0.92Control
5.30 ± 0.72 6.82 ± 2.02 *1.61 ± 0.53
15
1.29
0
Control
4.58 ± 1.53
Infected
6.13 ± 1.14 5.29 ± 0.63
2.47 ± 0.69
60 8
1.00
0.97
Control
6.10 ± 1.03 8.64 ± 1.80 *2.54 ± 0.81
Infected
3.14 ± 1.16
60
1.31
1
Infected
6.82 ± 2.02 *4.25± 0.92Control
6.59
±
0.80
3.05
±
1.93
15
1.29
0.93
Control
5.29 ± 0.63
4.58 ± 1.53
tion (h)After Infection (h)28Contrast%ID/g indicates percent injected dose per gram of tissue. * p < 0.01 vs. control muscle at ab
Infected
8.64 ± 1.80 *3.14 ± 1.16
1.31
1.03
after60infection.
Control
6.59 ± 0.80
3.05 ± 1.93
%ID/g indicates percent injected dose per gram of tissue. * p < 0.01 vs. control muscle at about 8 h after infection.

3.6. Planar Imaging with 123I-BMIPP in E. coli EC-14 Infection Model Mice
3.6. Planar Imaging with 123 I-BMIPP in E. coli EC-14 Infection Model Mice123

Figure 5 shows the planar images acquired 1.5
h after I-BMIPP injection an
Figure 5 shows the planar images acquired 1.5 h after 123 I-BMIPP injection and 9.5 h
after
infection with E. coli EC-14. The infected area was visualized, and the contra
after infection with E. coli EC-14. The infected area was visualized, and the contrast wasapproximately
1.33
times
higherthanofthatof thearea.
control area.
approximately 1.33
times
higherthan thatthe control

Figure 5. Imaging with 123 I-BMIPP in E. coli EC-14 infection model mice. With lead shielding

Figure 5. Imaging with 123I-BMIPP in E. coli EC-14 infection model mice. With lead shieldin
head to bladder, planar images were acquired for 5 min (1 frame) at 1.5 h after 123I-BMIPP
istration and 9.5 h after EC-14 infection. The infected muscle (arrow of right legs) is visualiz
the contrast is approximately 1.33 times higher than that of the control muscle (left legs).

Pharmaceutics 2022, 14, 1008

8 of 10from head to bladder, planar images were acquired for 5 min (1 frame) at 1.5 h after 123 I-BMIPP
administration and 9.5 h after EC-14 infection. The infected muscle (arrow of right legs) is visualized,
and the contrast is approximately 1.33 times higher than that of the control muscle (left legs).

4. Discussion
In this study, we investigated bacterial imaging using a radiopharmaceutical in common clinical use for fatty acid metabolism scintigraphy. Since fatty acids are components ofthe bacterial cell membrane [9,10], we explored the use of 123 I-BMIPP, a long-chain fatty
acid analog used for myocardial fatty acid metabolism scintigraphy in Japan [11–14], forbacterial imaging. 123 I-BMIPP exhibited marked accumulation in E. coli EC-14 during
the early growth phase (Figure 1), similar to the accumulation of 18 F-FDG (Table 1). This
specific accumulation suggests that EC-14 actively metabolizes fatty acids. In addition, theaccumulation of 123 I-BMIPP in EC-14 was higher than that of 18 F-FDG at the culture timeof 1h, whereas it was lower at the culture times of 3 and 6 h because glucose concentration
was regulated to 0.1 mg/mL in the culture medium for in vitro study with 18 F-FDG, andthe accumulation of 18 F-FDG increased the most at the culture time of 3 h.
The mechanism of 123 I-BMIPP accumulation in E. coli EC-14 was elucidated under theconditions of low temperature (Figure 2) and incubation in the presence of SSO, a CD36
inhibitor (Figure 3). In our group’s study, we have previously confirmed the inhibitory
effect of SSO on the uptake of BMIPP in cancer cells (data not shown). Therefore, SSO wasused in this study because it might also be relevant to bacterial accumulation. The rateof accumulation in EC-14 under low-temperature conditions was significantly lower thanthat of the control incubated at 37 ◦ C, potentially due to a reduction in bacterial metabolic
activity at the lower temperature, thereby impeding the uptake of 123 I-BMIPP. This result
indicates that the accumulation of 123 I-BMIPP in EC-14 depends on both bacterial growthand metabolic activity. In the presence of SSO, an inhibitor of the fatty acid transport
membrane protein CD36 [15,16], the accumulation of 123 I-BMIPP was significantly reduced
at 3 and 6 h of incubation compared with the control. This suggests that the mechanismof 123 I-BMIPP uptake by EC-14 is sensitive to SSO and involves a fatty acid transport
membrane protein such as CD36, which is present in human cells. CD36 binds longchain fatty acids and promotes their transport into cells [17]. The lack of significanceof the difference between the control and SSO treatment in the present study at 1 h ofincubation was possibly due to a large measurement error. In addition, during the earlystage of bacterial growth, other uptake mechanisms and/or metabolic activity could play asignificant role in uptake. As the incubation time increased, the effect of SSO became morenotable, which suggests that the bacterial uptake of 123 I-BMIPP is sensitive to transporter
selection. In addition, during the early stage of bacterial growth, other uptake mechanisms
and/or metabolic activity could play a significant role in uptake. As the incubation timeincreased, the effect of SSO became more notable, which suggests that the bacterial uptake of
123 I-BMIPP is sensitive to transporter selection. In addition, SSO inhibits the mitochondrial
respiratory chain, and reduced mitochondrial activity may reduce 123 I-BMIPP uptake. Thisis particularly important at later time points where significance is shown. The possibility
that 123 I-BMIPP is also taken up by bacteria through other pathways such as endocytosis
cannot be excluded. However, we assume that the CD36-like uptake mechanism is at least
one of the mechanisms of 123 I-BMIPP uptake by bacteria.
Biological distribution was examined in the E. coli EC-14 infection model mice using
123 I-BMIPP (Table 2) and 18 F-FDG (Table 3). Compared with 18 F-FDG, the accumulationof 123 I-BMIPP in the blood, lung, liver, and kidney was higher at 2 and 8 h post-infection,
indicating that 123 I-BMIPP has a longer retention time than 18 F-FDG in the primary organs.
As for the blood clearance result, the lack of clearance from blood was likely affected byproteins in the mouse body. Studies have shown that serum albumin plays a role in binding
and transporting fatty acids in the blood [18]. 123 I-BMIPP, a derivative of fatty acid, binds toalbumin in the mouse body, causing its retention in the blood. This may have resulted in the
delayed excretion of 123 I-BMIPP in this study. Moreover, in the in vivo experiment, the mice
were fully anesthetized and not moving at about 2 h post-infection, but at 8 h post-infection,

Pharmaceutics 2022, 14, 1008

9 of 10the mice were physically active, and the radioactivity in the blood was thought to have beentransferred to the myocardium. An increased myocardial accumulation of 18 F-FDG after
exercise has been reported [19]. One can assume that this may have led to the increased
18 F-FDG uptake in the heart. In addition, 123 I-BMIPP accumulation was higher than thatof 18 F-FDG in non-infected control muscle (Table 4) because 123 I-BMIPP may have had
a greater impact on walking and/or running than 18 F-FDG during the time between theinfection and sacrifice of the mice. 123 I-BMIPP exhibited a tendency toward greater imaging
contrast at both 2 and 8 h after infection compared with 18 F-FDG. A significant increasein accumulation in the infection area was observed at 8 h after infection (Table 4). Since
123 I-BMIPP accumulation reportedly decreases in myocarditis [20], it can be inferred that
123 I-BMIPP accumulated in the bacteria rather than in the inflamed tissues in this study.
123 I-BMIPP imaging in the E. coli EC-14 infection model mice was performed withlead shielding from head to bladder (Figure 5). In this study, the right leg of the mouse was
the E. coli-infected side, and the left leg was the non-infected side (control); the contrast
indicates the ratio of the infected area to the contralateral normal area. Thus, the imageof one mouse shows both infected and non-infected sides at the same time. The infectedmuscle imaged 1.5 h after 123 I-BMIPP injection and 9.5 h after infection exhibited higher
contrast than the non-infected control muscle. The infected area, as determined by the
123 I-BMIPP signal, was approximately 1.33 times larger than the 123 I-BMIPP control area.
Analysis of the biodistribution of 123 I-BMIPP 8 h after infection (Table 4) showed that theratio of 123 I-BMIPP accumulation in the infected muscle to that in control muscle was 1.29 at
15 min and 1.31 at 60 min after 123 I-BMIPP injection. Thus, the lead shield had a negligible
effect on planar imaging.
Clinical SPECT imaging in humans may show less 123 I-BMIPP accumulation in control
muscle than imaging in mice, because humans can rest before SPECT imaging. This may
result in a higher image contrast at the infection site, making infected areas more clearlyvisible in clinical SPECT imaging.
As a limitation of this study, there may be difficulty when using 123 I-BMIPP to detectbacterial infections in the trunk of the body, because of its accumulation and retention,
but it could be very useful in infections involving the upper and lower limbs. 123 I-BMIPP
accumulates in normal muscle, but it has better contrast ratios than 18 F-FDG at sites ofbacterial infection (Table 4).
5. Conclusions
123 I-BMIPP can take up E. coli EC-14 via a fatty acid transport membrane protein suchas CD36 and accumulate by fatty acid metabolism. 123 I-BMIPP has the potential to visualize
fatty acid metabolism in bacteria for bacterial infection imaging, especially in upper andlower limbs.
Author Contributions: Conceptualization, Y.M., M.K. and K.K.; methodology, K.N. (Kodai Nishi),N.S., K.O., F.T. and S.O.; investigation, Y.M., A.M., K.N. (Koya Nakamoto), K.O. and M.M.; resources,
H.M.; writing—original draft preparation, Y.M.; writing—review and editing, K.Y., R.N., M.K. andK.K.; supervision, H.M. All authors have read and agreed to the published version of the manuscript.
Funding: This study was funded in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 18K19508, No. 18K07747 and No. 21H02865) and SHIONOGI &Co., Ltd.
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Review Board of Kanazawa University (protocol code AP-183983 and date of approval 26 March 2021).
Informed Consent Statement: Not applicable.
Acknowledgments: The authors would like to thank Mikie Ohtaki, Shusei Ohata, Takeaki Kiritani,
Rika Terada, Yoshie Yuasa, Akane Yonezawa and other staff of the Faculty of Health Sciences,
Kanazawa University.

Pharmaceutics 2022, 14, 1008

10 of 10

Conflicts of Interest: Fumika Takagi, Kenichi Okazaki and Hideki Maki are employees of Shionogi& Co., Ltd. The company had no role in the design of the study; in the collection, analyses, orinterpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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